Methanotrophic potential of Dutch canal wall biofilms is driven by Methylomonadaceae

Abstract Global urbanization of waterways over the past millennium has influenced microbial communities in these aquatic ecosystems. Increased nutrient inputs have turned most urban waters into net sources of the greenhouse gases carbon dioxide (CO2) and methane (CH4). Here, canal walls of five Dutch cities were studied for their biofilm CH4 oxidation potential, alongside field observations of water chemistry, and CO2 and CH4 emissions. Three cities showed canal wall biofilms with relatively high biological CH4 oxidation potential up to 0.48 mmol gDW−1 d−1, whereas the other two cities showed no oxidation potential. Salinity was identified as the main driver of biofilm bacterial community composition. Crenothrix and Methyloglobulus methanotrophs were observed in CH4-oxidizing biofilms. We show that microbial oxidation in canal biofilms is widespread and is likely driven by the same taxa found across cities with distinctly different canal water chemistry. The oxidation potential of the biofilms was not correlated with the amount of CH4 emitted but was related to the presence or absence of methanotrophs in the biofilms. This was controlled by whether there was enough CH4 present to sustain a methanotrophic community. These results demonstrate that canal wall biofilms can directly contribute to the mitigation of greenhouse gases from urban canals.


Introduction
Urban waters are increasingly recognized as important ecosystems that contribute significantly to global greenhouse gas emissions.As e v en low methane (CH 4 ) emissions can hav e a gr eat impact on climate warming (Myhre et al. 2013 ), CH 4 with its high global warming potential has been the focus of se v er al studies of urban ponds (van Bergen et al. 2019, Peacock et al. 2021 ) and even whole cities (Martinez-Cruz et al. 2017, Herr er o Ortega et al. 2019, Wang et al. 2021 ).While str eams, riv ers, and lakes are well studied (Bastvik en et al. 2011, Stanle y et al. 2016, Saunois et al. 2020 ), urban canals and ditc hes ar e poorl y r epr esented in r ecent datasets (Peacock et al. 2021 ).
Greenhouse gas emissions from aquatic systems are the result of microbial respiration and anaerobic degradation of organic matter (Conrad 2009, Dean et al. 2018 ).Urban canals are susceptible to many factors that could increase anoxia, and, consequently, higher CH 4 fluxes, such as slo w, laminar w ater flo w and high levels of nutrients (Needelman et al. 2007, Peacock et al. 2021 ).Lo w er availability of oxygen causes a larger methanogenic zone and increases CH 4 emissions.In the Netherlands, many urban waters ar e consider ed to hav e lo w w ater quality due to phosphate and ammonia loading, yearly algal blooms, and excessive human acti vity lik e boating and r ecr eation (Teurlincx et al. 2019, Armstr ong et al. 2022 ).Ther efor e, urban canals hav e the potential to be a substantial source of CH 4 in the Netherlands (Stanley et al. 2016, Peacock et al. 2021 ).Due to the extensive use of canals in many Dutch cities , the en vir onmental impact might be consider able but has been poorly constrained.
Methanotrophic bacteria (MOB) and archaea in the sediment or water column can consume CH 4 , acting as a biological filter.In the sediment, anaerobic CH 4 oxidation (AOM) can occur in freshwaters using a variety of electron acceptors such as NO 3 − , NO 2 − , ir on, and or ganic matter (Ettwig et al. 2010, Haroon et al. 2013, Ettwig et al. 2016, Valenzuela et al. 2019 ).For se v er al lakes and str eams, this AOM pr ocess has been observed to be ecologically r ele v ant (Martinez-Cruz et al. 2018, Shen et al. 2019 ).The aerobic MOB Bacillus methanica was in fact first isolated from an urban canal in Delft by Söhngen ( 1906 ), and aerobic methanotrophs are considered to be an important CH 4 sink at the global scale due to their high CH 4 conversion rates (Frenzel et al. 1990, Hanson and Hanson 1996, Knief 2015 ).In lakes and riv ers, MOB thriv e at the sediment-water interface or in the water column.Se v er al studies have determined the methanotrophic potential of the water column in lakes ( e.g ., Carini et al. 2005, Guérin and Abril 2007, Thottathil et al. 2019, Reis et al. 2020 ), but for flowing waters, this has been performed only for the rivers Saar and Elbe, Germany (Zaiss et al. 1982, Matouš ů et al. 2017 ), the Yellow River, China (Hao et al. 2020 ), and the Condamine Riv er, Austr alia (Burr ows et al. 2021 ).
Recently, a study by Pelsma et al. ( 2022 ) reported a novel urban habitat for MOB in the form of the canal wall biofilm.Exposed to both air and water, these biofilms were hypothesized to be an excellent habitat for methanotrophs as they experience little water turbulence and are exposed to high CH 4 concentrations.The biofilm methanotroph present w as Meth yloglobulus morosus , pr e viousl y isolated fr om lake sediment (Deutzmann et al. 2014 ).Ho w e v er, little is known about methanotrophic biofilms in the built aquatic environment.We hypothesized that MOB are present and metabolicall y activ e in a br oader r ange of urban aquatic systems.To test this hypothesis, we sampled canal wall biofilms in five cities in the Netherlands with canals ranging from saline to freshw ater.In addition, w e investigated the impact of surface material on the micr obial comm unity and methanotrophic activity.Lastly, we synthesized field observations and flux data to better identify potential urban hotspots of microbial CH 4 cycling.

City selection and sample sites
The Netherlands has many cities with urban canals as part of the citysca pe.We c hose fiv e r epr esentativ e cities along a salinity gradient with different environmental parameters in order to capture se v er al types of biofilms in this cross-sectional study.The city of Middelburg was chosen as it is known to be saline due to its proximity to the Scheldt estuary and North Sea.Den Helder and Zwolle wer e c hosen based on their location in the northern and eastern extremities of the Netherlands.Zaandam, like Amsterdam, is influenced by the saline Noordzeekanaal and has br ac kish canals.Leiden was chosen to represent a freshwater canal system in the central Netherlands.All cities were sampled betw een Mar ch and July 2022, with a minimum of four sample sites per city (Table 1 , Fig. 1 ; https://methanecanals.shinya pps.io/data _ ma ps/).Sample sites within each city were chosen based on the accessibility to the air-water interface of the canal wall from the street level, whilst also allowing for a dispersed spread of locations.Sampling was undertaken on a single day for each city and samples were processed to a stable state within 24 h after sampling.Maps with sampling sites and photogr a phs of the locations ar e pr esented in the Supplementary Materials .

In situ flux measurements and canal water chemical analyses
Diffusive fluxes of CH 4 and carbon dioxide (CO 2 ) were measured using the floating chamber method (Lorke et al. 2015 ) and a portable greenhouse gas analyser (LI-7810 CH 4 /CO 2 /H 2 O Trace Gas Analyser; LI-COR Inc., USA).Triplicate measurements were done at each site and measurements influenced by gas bubbles w ere discar ded from the analysis.A bubble event was recognized either by eye or when a very sudden CH 4 spike was detected that w ent upw ar ds of 5000 ppb.Fluxes w er e calculated thr ough linear r egr ession of the CH 4 or CO 2 concentration over the measurement time (Van Bergen et al. 2019 ): In situ pH, conductivity, temper atur e, and dissolv ed oxygen wer e measur ed using a Hach HQ4300 m ulti-par ameter pr obe (Hach, The Netherlands).Canal water concentrations of NO 3 − , NH 4 + , and PO 4 3 − were measured using colorimetric assays on an AutoAnal yzer3 (Br an + Luebbe, German y) after filtering 40 ml water using a nylon 0.22 μm syringe filter into a sterile 50 ml centrifuge tube.Samples were stored at −20 • C if they were not measured within 1 week.Total organic carbon (TOC) and total nitr ogen wer e measur ed on a TOC-L CPH/CPN analyser (Shimadzu Benelux, The Netherlands) using unfiltered canal water.Metal concentrations of Cr, Mn, F e , Co, Ni, Cu, Zn, As , Se , Sr, Mo, Cd, Ba, La, Ce, Nd, Hg, and Pb were measured by inductively coupled plasma mass spectrometry on an Xseries-I (Thermo-Fisher Scientific, Germany).For metals, 10 ml of canal water was acidified with 1% nitric acid (Supr a pur, Merc k, German y) to pH ∼2 prior to analysis.

Biofilm sampling
At each site, three biofilms were scraped from the surroundings and collected in sterile 50 ml centrifuge tubes.Scr a ping was done using a disposable metal razor that was disinfected using 70% ethanol and air-dried before scraping.For each biofilm subsample, a new razor was used to prevent cross-contamination.Accessibility to the canal wall pr e v ented sampling in some locations, so another static object with visible biofilm gro wth w as sampled instead like a wooden pole or floating pontoons in a harbour.For comparison to the w ater column, w ater samples w ere taken in autoclaved glass bottles by rinsing them with canal water three times and sealing the bottles under water.

Micr ocosm CH 4 o xida tion incuba tions
Oxidation rates of CH 4 were determined in triplicate per biofilm subsample and were started within 24 h after sampling.Biofilms wer e distributed equall y in autoclav ed 120 ml glass serum bottles.Forty millilitre AMS medium (0. and 0.717 g l −1 Na 2 HPO 4 •12H 2 O; made in Milli-Q water and autoclaved before adding the phosphate buffer; Whittenbury et al. 1970 ) supplemented with SL-10 trace elements (German Collection of Microorganisms and Cell Cultures) was used for all biofilm incubations.Additionall y, to suppl y XoxF-type methanol dehydrogenases with lanthanide metals (Keltjens et al. 2014 ), LaCl 3 was added to a final concentration of 2 μM.Bottles were capped with grey rubber stoppers and sealed with aluminium crimp caps.While these grey rubber stoppers are less thick than red butyl rubber stoppers, they do not have the potential to inhibit pmoA-based methanotrophy (Niemann et al. 2015 ).For the canal water incubations, 40 ml of the canal water was used per replicate without any amendments and capped in the same way as the biofilm incubations.Abiotic contr ols of eac h biofilm subsample wer e measur ed in duplicate by autoclaving the sealed bottles.
A volume of 5 ml of CH 4 was added to each bottle, after which the bottles were brought up to an overpressure of 0.25 bar by injecting lab air through a 0.22 μm cellulose acetate membrane filters, amounting to a final headspace CH 4 concentration of 5%.Pr essur es wer e c hec ked using a digital pr essur e meter (GMH 3111, GHM Messtec hnik GmbH, German y).Prior to the first measur ement, bottles were left to equilibrate for 3 h at room temperature (21 • C).Headspace CH 4 concentrations were measured daily for at least 2 weeks using 50 μl injections on an HP 5890 Series II (Agilent Tec hnologies, USA) gas c hr omatogr a ph equipped with a Por a pak Q column (80/100 mesh) and a flame ionization detector.Headspace pr essur es wer e measur ed after eac h measur ement day.Using a calibr ation curv e, headspace CH 4 concentr ations wer e calculated and adjusted using the measured pressures.Oxidation rates were calculated as the slope of a linear r egr ession fitted to data points of the first eight days of incubation.Within this timeframe, biofilm incubations could be considered linear (median R 2 of 0.9).For comparison, biofilms were normalized to gram dry weight (g DW ) by drying out 10 ml of well-mixed biofilm incubation medium in an 80 • C sto ve .
To determine whether a CH 4 oxidation rate was due to biological activity or due to other physical effects, all rates were

DN A extr action and 16S rRN A gene amplicon sequencing
Biofilm DN A w as extracted using the DNeasy Po w erSoil DN A extr action kit (Qia gen, The Netherlands) by weighing ∼300 mg in a Po w erBead tube.The manufacturer's instructions were followed except for the homogenization step as this was done using a Tis-sueLyser LT (Qiagen) at 50 Hz for 10 min.DN A w as eluted in diethyl pyr ocarbonate-tr eated water (Invitr ogen, USA).Eluted DNA was stored at −20

Sequencing data analysis
Raw sequencing reads were filtered and called to amplicon sequence variants (ASVs) using the package dada2 (Callahan et al. 2016 ;v1.26)

Sta tistical anal yses
Comparison of CH 4 oxidation rates to the abiotic control was done using the permuted non-parametric Brunner-Munzel test available in the R package brunnermunzel (Neubert andBrunner 2007 , Ara 2022 ).The test was single tailed with a significance of 5% ( α = 0.05).Comparison of the biofilm surface type and oxidation potential was done using the Mann-Whitney U test through the function wilcox_test available in the R package rstatix (Kassambar a 2023 ).Corr elation anal yses on envir onmental v ariables were done using Kendall's τ coefficient.Ordination using non-metric multidimensional scaling (NMDS) was done using the function metaMDS from the package vegan (Oksanen et al. 2022 , v2.6.4).Envir onmental par ameters wer e tested for significance and fitted to the ordination using envfit .All environmental parameters sho wn w ere statistically significant after testing with 999 permutations ( p < .001).

Physico-chemical properties of se ver al Dutch urban canals
Five cities with urban canals were sampled to determine the potential of the canal wall biofilm to consume CH 4 .The canals sho w ed large differences in salinity, with Middelburg canals being highly saline, while Leiden and Zwolle had freshwater canals.Zaandam and Den Helder were in between freshwater and br ac kish canals ( Table S1 ).The canal water temper atur e incr eased fr om Zwolle (6 • C) to Zaandam (20 • C) because of the time of year during sampling.Nutrient concentrations differed between cities, with freshwater canals in Zwolle, Den Helder, and Leiden containing increased le v els of NO 3 − and NH 4 + up to 90 and 25 μmol l −1 , r espectiv el y.
In contrast, the saline canals of Middelburg contained little NO 3 − (0.13-3.53 μmol l −1 ) and NH 4 + ( ∼3.5 μmol l −1 ).The highest concentrations of PO 4 3 − were measured for Den Helder and Zaandam (5.4-29.7 μmol l −1 ), whereas in the other cities, concentrations w ere belo w 1 μmol l −1 .Total organic carbon v alues r anged fr om 5.7 to 15.5 mg l −1 ( Table S1 ), indicating incr eased or ganic carbon loading in the canals, especially in the city of Leiden.The highest iron and cerium concentrations were measured in Zwolle, specifically at site 1 with the lo w est pH of 7.36, at 2.98 mg l −1 .Copper concentr ations wer e highest in Middelbur g (saline) with 6.93 μg l −1 and Leiden (freshwater) with 9.37 μg l −1 .Dissolved oxygen was alwa ys abo ve 50% air saturation, indicating that anoxia was not occurring in the water columns at the sampling sites.Diffusiv e flux measur ements of CH 4 and CO 2 sho w ed that e v ery city except Middelburg could be considered a consistent source of CH 4 .For Zaandam and Zwolle, the observed CH 4 flux was highest at 1.8 and 0.69 mmol m −2 d −1 , r espectiv el y (Fig. 2 ).Both Zwolle's and Zaandam's canals were a source of CO 2 as well at median flux of 158 and 51 mmol m −2 d −1 , r espectiv el y.For the saline canals of Middelbur g, CH 4 emissions wer e onl y detectable for site 5 (0.14 mmol m −2 d −1 , thr ee r eplicate measur ements), wher eas the CO 2 flux was mostly negative except for site 5 (av er a ge of 11.79 mmol m −2 d −1 ).Despite high nitrate and TOC, the canals in Leiden did not emit CO 2 and median CH 4 emissions wer e onl y 0.18 mmol m −2 d −1 .Canals in Den Helder, like Zaandam, were a net source of both CH 4 and CO 2 but with some spatial variability within the city.

CH 4 oxidation on the canal wall is driven by Methylomonadaceae
Den Helder, Zaandam, and Zwolle hosted canal wall biofilms with clear biological CH 4 r emov al (Fig. 3 ).Ho w e v er, se v er al biofilms, for example, sites 1-5 in Middelburg and sites 2-5 in Leiden, sho w ed CH 4 oxidation rates of < 0.1 mmol g DW −1 d −1 , which were not significantl y differ ent fr om the contr ol incubations .T he highest observed CH 4 oxidation rates were 0.48 mmol g DW −1 d −1 in Zwolle (March), 0.31 mmol g DW −1 d −1 in Den Helder (May), and 0.12 mmol g DW −1 d −1 in Zaandam (J uly).T he variability between biofilm subsamples was high as indicated by the large interquartile range (0.01-0.35 mmol g DW −1 d −1 in Zwolle) for the biofilm incubations (Fig. 3 ).Water column methanotrophy was observed in all cities except Zwolle but with low statistical significance ( Fig. S1 ).Biofilm bacterial community profiling sho w ed a dominance of the gamma pr oteobacterial methanotr ophic species Crenothrix and Methyloglobulus (Fig. 4 ).Most sampled canal walls were made of either wood or brick.Depending on the city, methanotrophs were more abundant on brick than on wood, but no clear pattern was observed between abundance and canal wall material.In Middelbur g, no methanotr ophy was observ ed and the sequencing r esults indeed sho w ed no reads classified as either Crenothrix or Methyloglobulus .In Zaandam, these species were observed but only at a r elativ e abundance of 2%, whic h corr esponds quite well to the lo w er CH 4 oxidation rates (Fig. 3 ).
Analysis of the total bacterial community revealed a strong pattern based on salinity and nutrient le v els (Fig. 5 ).High NH 4 + and Fe concentrations in Zwolle clustered it together with Figure 3. Biofilm methanotrophic rates for each sampling site, ordered by city.Comparison against the abiotic control was done using a Brunner-Munzel test.Per site, nine biofilm incubations were compared against six control incubations.Biofilm incubations with statistically significant differences from the control are marked by an asterisk.* p < .05,* * < .01,and * * * p < .001.

Regulators of CH 4 emissions
The potential of urban canals to emit significant amounts of CH 4 has been well documented in recent years (Stanley et al. 2016, Peacock et al. 2021, Rosentreter et al. 2021, Wang et al. 2021 ) and the results obtained here support this.Out of the five sampled cities, only the saline canals in Middelburg were not a source of CH 4 .
As the canals of Middelburg contained the least amount of dissolved nutrients and were fully o xygenated, the y can be considered a marine system.Ho w ever, during the fieldw ork, w e observed turbid water with a lot of algal growth in these canals.Due to the time of year we sampled in Middelburg (early April), algal death in late summer could contribute to CH 4 production and emission in other seasons.
There was some spatial separation of CH 4 fluxes within the Zwolle city centre, with site 1 having the highest CH 4 emissions (Fig. 2 ).This site was c har acterized by a shallow waterway, so the increase in emissions could be explained by a more turbulent water column that r esuspends sediment, likel y also causing high dissolved Fe concentrations at this site.Ho w ever, the other sites were subject to boating, leading to similar resuspension of canal sediment.The NH 4 + and NO 3 − concentr ations measur ed in these canals could contribute to eutrophication of the canal sys-tem and subsequent increases in sediment methanogenesis.With our sampling date for Zwolle in early spring, these canals could have higher CH 4 emissions in late summer not captured by our study.The other freshwater city, Leiden, had substantially lo w er NH 4 + concentrations and CH 4 emissions (Fig. 2 , Table S1 ), but higher PO 4 3 − concentrations .T he sediment floor of these canals w as dominated b y plants, which could help in aerating the sediment and thereby decreasing methanogenesis in the sediment (Bastviken et al. 2023 ).

Biofilm methanotrophy is present in di v erse urban canals
The city of Zwolle harboured biofilms with up to 8% methanotrophs, and biological CH 4 consumption was found at all sites.
Compared to the Amsterdam data, we detected similar species but at higher r elativ e abundances and oxidation rates .T he absence of methanotrophs from all sampled biofilms in Middelburg in April is consistent with its very low CH 4 oxidation potential.
Ho w e v er, the incubation medium used in this study was not optimized for saline conditions and could have affected the observ ed methanotr ophic r ate.Similarl y, it has been documented that in the nearby Lake Gr e v elingen, the r elativ e abundance of methanotrophs and CH 4 oxidation potential are also lo w est around the end of March (Egger et al. 2016, Venetz et al. 2022, Żygadłowska et al. 2023 ).Only one sampled biofilm from Leiden consumed CH 4 , suggesting it is unlikely that Leiden's biofilms host methanotrophs.Biofilm incubations from Zaandam sho w ed that both sites 1 and 2 did not consume CH 4 and only site 2 harboured Methylomonadaceae methanotrophs .T hese sites ar e spatiall y separ ated fr om eac h other by one sluice gate (pictured in Fig. 1 b).During the fieldwork, the sluice gate was opened causing a CH 4 ebullition e v ent.If most CH 4 is emitted through bubbles, MOB do not have a constant supply of substrate and could be outcompeted by other bacteria in the biofilm.For the other two sites in Zaandam, no ebullition was observed but biofilms did oxidize CH 4 and harbour ed methanotr ophs, meaning a consistent, if lo w er, CH 4 supply is sufficient to sustain a biofilm community with methanotrophic potential.Den Helder was very similar to Zaandam in terms of water chemistry but had much higher oxidation rates.Ho w ever, for Den Helder site 1, only one subsample had a r elativ e abundance of 9% Methylomonadaceae , corresponding to the highest rate.For site 3, no CH 4 oxidation rate was measured and no MOB could be detected with sequencing (Fig. 4 ).This site was a small domestic harbour very similar to the other four sites .T her efor e, it is e vident that within a city canal network, there are local effects of which our study could only capture the heterogeneity to a certain extent.Futur e r esearc h is ad vised to tak e spatial heterogeneity into account during study design, as day-night cycles, point sources, and local infr astructur e (like sluice gates) can have great effects on both the microbial community and CH 4 dynamics (Attermeyer et al. 2021, Stanley et al. 2022 ).

Conclusions
Our data indicate the presence of biofilm methanotrophy in several distinct urban canal networks across the Netherlands .T he two dominant methanotrophs were Crenothrix and Methyloglobulus , in line with what was pr e viousl y found in canal biofilms in Amsterdam.The presence of these methanotrophs was associated with consistent diffusive CH 4 emissions .T heir presence could be a way to identify canals with high CH 4 -cycling activity.CH 4 emissions wer e observ ed fr om most canals and could contribute substantially to total urban CH 4 emissions.NH 4 + concentration was positively correlated with CH 4 emissions and CH 4 oxidation rate, while salinity correlated negatively.We sho w ed that canal wall biofilms may assist in mitigating CH 4 emissions across a diverse range of Dutch cities.A widespread, year-round monitoring of CH 4 emissions (diffusive and ebullitive) from urban canals, in conjunction with microbial activity experiments, is required for a better understanding of these ubiquitous and unique Dutch landmarks.

Figure 1 .
Figure 1.Location of the five sampled cities within the Netherlands (A).Examples of canals in Zaandam (B) and Middelburg (C).Sampled biofilms in Zwolle (F and D) and Leiden (E) showed that there is great diversity in the types of canals and biofilm growth surfaces.An interactive version of a sample site map is available at https://methanecanals .shinyapps.io/data _ maps/ .
in R4.1.2(R Core Team 2022 ).ASVs were assigned taxonomy based on the SILVA SSU 138.1 database(Quast et al. 2012 ).After trimming, denoizing, dereplication, and c himer a r emov al, each sequencing sample had a minimum 24 103 merged reads with a maximum of 67 592.Biofilm r aw r eads fr om the Amsterdam 2019 dataset(Pelsma et al. 2022 ; PRJEB40426)  were re-analysed with the updated pac ka ges and added to the analysis.Using the phyloseq pac ka ge (McMurdie and Holmes 2013 , v1.42.0), sequence abundance tables were converted to R dataframes.Graphs were constructed using pac ka ges av ailable in the pac ka ge libr ary tid yverse(Wickham et al. 2019  ).

Figure 2 .
Figure 2. Diffusive flux measurements for CH 4 (left panel) and CO 2 (right panel) for the five investigated cities.All individual data points are shown and coloured by sample sites within the city.Note the different y -axes for each gas.

Figure 4 .
Figure 4. Relative abundances of total bacterial 16S rRNA gene reads of known methanotrophic genera for six sequenced cities. Bars are coloured by sampling site within each city.Leiden biofilms were not sequenced due to DNA extraction difficulties, which is indicated by a blank column.The Amsterdam dataset was obtained from a previous study(Pelsma et al. 2022 ).Note the different scales for the Zwolle samples.

F igure 5 .
Or dination of the total bacterial community based on 16S rRNA gene amplicon sequencing with Bray-Curtis dissimilarity as distance metric.Envir onmental v ariables ar e scaled based on their effect.Amsterdam (data from Pelsma et al. 2022 ), while Den Helder and Zaandam also grouped together, separately.Despite differences in NO 3 − concentrations, it did not explain the differences in biofilm community composition as significantly as CH 4 emissions and PO 4 3 − .The single br ac kish site in Middelbur g formed a grouping closer to Den Helder and Zaandam, suggesting a stronger influence of salinity than nutrient le v els on the biofilm microbial community.

Table 1 .
Sample locations within the cities.
Munzel test.Only when the median oxidation rate of the biofilms was statistically significant from the abiotic controls ( P < .05)did we report that a canal wall biofilm can oxidize CH 4 .